The invention relates to DNA sequences coding for sterol glycosyl transferases as well as the use thereof to modify the content and/or the structure of sterol glycosides and/or their synthetic secondary products in transgenic organisms. Sterol glycosides and the biosynthetic secondary products steryl oligoglycosides and acylated sterol glycosides are natural substances found in plants as well as in some fungi and bacteria. For these substances and their secondary products a variety of physiological effects have been described such as for example inhibition of the vascular permeability, anti tumor activity antiphlogistic and haemostatic effect (Okuyama, E and Yamazaki, M (1983) Yakugaku Zasshi 103: 43 ff; Normura, T.; Watanabe, M.; Inoue, K. and Ohata, K. (1978) Japan J. Pharmacol. 28, Suppl. 110 P; Miles, D. H.; Stagg, D. D. and Parish, E. J. (1979) J. Nat. Prod: 42: 700 ff; King, M. L.; Ling, H. C.; Wang, C. T. and Su, M. (1979) J. Nat. Prod. 42: 701 ff.; Seki, J.; Okita, A.; Watanabe, M.; Nakagawa, T.; Honda, K.; Tatewaki, N. and Sugiyama, M. (1985) J. Pharm. Sci. 74: 1259-1264), which suggest an application as therapeutically effective substances for human beings. So far only xcex2-sitosterol-xcex2-D-glycoside, which is isolated from plants, can be bought as a medication for the treatment of prostrade hyperplasis (for example as bloom oil capsules, Hoyer Ltd., Neuss). A disadvantage of the substances lies in the fact that they exist in the organisms in only relatively small amounts and that they have to be extracted and purified by highly expensive methods. Furthermore, some of the organisms, which contain these substances are human-pathogenic and can only be cultivated with a high expenditure which makes their potential use as medication, detergents, emulgators, as basic material for synthetic materials and for the production of liposomes when needed in large amounts and of higher purity, fairly inapplicable at this point in time.
The enzymatic synthesis of sterol glycosides in the organisms of sugar nucleotides and sterols with a free OH-group is catalyzed by the sterol glycosyl transferases (in short: sterol glycosyl transferases) which are dependent on sugar nucleotides. These enzymes can be partly isolated and purified from the organisms, but are not available for economic use in sufficient quantities and qualities.
The activity of these enzymes can be proven with special in vitro enzyme detection systems. Furthermore, in one particular case a sterol glycosyl transferases from oat could be purified to the point of homogeneity. (Warnecke and Heinz, 1994) so far, however, no gene or any other nucleic acids has been known which codes a sterol glycosyl transferases.
Furthermore some nucleic acid sequences are known, which are similar to the sequence described in this patent application. In no case however, a sterol glycosyl transferase activity of the matching transcription product has been shown for the same or has even been discussed. Such nucleic acid sequences can only be used to manipulate the content and/or the composition of sterol glycosides and secondary products in certain organisms and thereby positively modify relevant characteristics of such organisms. That way cultivated plants can be produced with a better tolerance or resistance against hazardous environmental influences such as saline soil, drought, cold and freeze. Also micro organisms as for example, baker and brewing yeast can be improved with regard to ethanol and temperature tolerance.
In addition to the reaction product sterol glycoside, the enzyme itself can be of economical use when it can be produced purely and in large quantity by the application of genetic engineering. An example for this is the use of cholesterol quantification.
Furthermore the sterol glycosyl transferasesxe2x80x94and the respectively coding DNA sequencesxe2x80x94based on their similarity of sollanidine with sterolsxe2x80x94can also be used as enzymes or the supply of such enzymes, which are responsible for the synthesis of solanine in solacene. This enables the production of plants, which are modified by genetic engineering, with low solanine or which are solanie free. By choosing the suitable methods such a reduction can be limited to certain parts of the plant or certain stages of development.
It is the task of the present invention to provide nucleic acid fragments with which transgenic organisms can be produced, which have improved economically relevant characteristics or with which in vivo or in vitro sterol glycosides and their secondary products can be produced
a) in larger quantities than in the original organisms; or
b) produced from organisms which are easier and simpler to cultivate than those in which these substances occur naturally; or
c) which are of a new structure and which have more favorable characteristics.
A method has been invented to control the synthesis of sterol glycosides and their secondary products. For this, nucleic acid fragments are provided which code sterol glycosyl transferases to produce chimerical genes. These chimerical genes can be used to transform cell cultures, plants, animals or micro organisms and thereby modify their sterol glycoside synthesis.
The invention relates to
(1) an isolated DNA fragment or recombinant DNA construct containing at least one part of a sequence coding sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense;
(2) a protein which derives from one nucleic acid sequence illustrated in FIGS. 1-3 or 11-22;
(3) plasmides, viruses or other vectors, which contain nucleic acid sequences as defined in (1);
(4) genomic clones containing genes or parts of genes which code a sequence as defined in (1);
(5) a chimerical gene which is able to modify the content of sterol glycosyl transferase or sterol glycosyl transferases in the strictest sense, especially sterol glycosyl transferase or sterol glycosyl transferases in the strictest sense;
(6) transformed cells, transformed micro organisms, plants or parts of plants containing a chimerical gene as defined in (5);
(7) a method for producing sterol glycoside entailing the cultivation of the transformed organisms defined in (6);
(8) the sterol glycosides or their secondary products obtained from the method defined in (7);
(9) a DNA fragment obtained according to one of the following methods or parts thereof:
a) use of one of nucleic acid sequences illustrated in FIGS. 1-3 or 11-13 or 17 as hybridization sample;
b) use of the amino acid sequences illustrated in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 for the synthesis of peptides or proteins which serve the obtaining of antisera; or
c)
i) comparing of the nucleotide sequences illustrated in FIGS. 1-3, 11-13 or 17 or the amino acid sequences derived thereof illustrated in FIGS. 4, 5, 14-16, 18, 19, 21 or 22 with each other or with already known nucleotide sequences or amino acid sequences derived thereof,
ii) deriving and syntethisingsizing of suitable specific oligonucleotides from similar areas of these sequences, and
iii) use of these oligonucleotides to produce nucleic acids coding for sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense especially for sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense or parts thereof with the help of a sequence depending protocol, especially the PCR method.
(10) a chimerical gene containing a DNA fragment defined in (9) and which is able to modify the content of sterol glycosyl transferase or sterol glycosyl transferase in the strictest sense especially sterol glycosyl transferase or sterol glycosyl transferase in the strictest sense in a transformed cell;
(11) transformed cells containing a chimerical gene as defined in (10);
(12) organisms, especially micro organisms such as bacteria and yeast whose gene or genes coding sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense, especially sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense, are deleted or interrupted by transformation with suitable chimerical genes.
(13) sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense, especially sterol glycosyl transferases or sterol glycosyl transferases in the strictest sense or parts thereof or fusion proteins with the already mentioned transferases which can be obtained from organisms as defined in (6) or (11) and
(14) antisera or products made of antisera, antibodies and parts thereof which are directed to a protein as defined in (13).
The nucleic acid fragments coding for sterol glycosyl transferases (FIGS. 2, 17) could be isolated from avena sativa and arabidopsis thalliana. The amino acid sequences derived from these nucleic acid sequences have a surprisingly low similarity to the already known sequences of steroid hormone glucoronosyl transferases. Therefore, it is quite surprising that we were able to isolate completely new nucleic acid fragments with our methods. So far it has not been possible to identify another nucleic acid fragment, which codes for sterol glycosyl transferases. The isolated eucaryotic nucleic acid fragments are characterized by the fact that they are surprisingly suited, fitted with respective control sequences, for effecting the synthesis of enzymatically active sterol glycosyl transferases in eucaryotic as well as in procaryotic organisms and within the same without the typically eucaryotic processing and modification.
The invention also relates to isolated nucleic acid fragments whose derived amino acid sequences have defined similarities to the derived amino acid sequences in FIG. 12 or 13. The invention also relates to all plasmides, viruses and other vectors which contain these isolated nucleic acid fragments or parts thereof.
The amino acid sequence illustrated in FIGS. 4 and 18 have remarkable similarities with the derived amino acid sequence of a genomic DNA piece from s. cerevisiae (see FIG. 9). Thereby dealing with the chromosome XII cosmid 9470 (gene bank no. gb U17246). The similarity is related to the 3xe2x80x2-range of the open reading structure of bp 32961-36557 (gene L9470.23). For this putative gene no function has been known so far. Several parts of this gene are provided with suitable control sequences and were able to prove sterol glycosyl transferases activities in cell homogenates of the transgenic cells after transformation of E. coli with this chimerical gene.
Furthermore, the invention also relates to the use of nucleic acid sequences of FIGS. 1-3, 11-13 and 17 or the amino acid sequence derived thereof for the isolation of genes or cDNAs coding for other sterol glycosyl transferases. This relates to the use of sequences or parts thereof as hybridization samples, use of antibodies against a polypeptide for example, which is coded by the nucleic acid fragments or derives thereof respectively. Furthermore the derivation of oligonucleotides and the use thereof in the PCR method from the nucleotide- or amino acid sequences is also effected by the comparison with other sequences.
The invention relates to all plasmides, viruses and other vectors containing the nucleic acid sequences from the FIGS. 1-3, 11-13, 17 or parts thereof or the yeast gene L9470.23 or parts thereof or nucleic acid fragments or parts thereof which were isolated according to the methods described in the foregoing paragraph and which are suited for expression of sterol glycosyl transferases in transformed cells. Patent is also claimed for all organisms (micro organisms, animals, plants, parts thereof, cell cultures) which contain these chimerical genes or the products and extracts thereof, if the substantial composition of these organisms has been modified by these chimerical genes.